Translocation of a polymer through a nanopore

ABSTRACT

Embodiments of the present disclosure are directed to methods, systems and devices, for analyzing the molecules. For example, in some embodiments, a system is provided which includes a first volume of conducting fluid, a second volume of conducting fluid, an orifice in communication with said first and second volumes of fluid, and means for applying an electric potential difference between said first and second volumes of fluid. In some such embodiments, a conjugate product is provided which comprises charged polymers each having attached thereto at least one first molecule for analysis, where the product carries a predetermined charge greater than the charge on the first molecule, and upon dissolving a product in the first volume of fluid, the product is directed into the orifice.

Embodiments disclosed in this application were made with governmentsupport under R01 HG006323 awarded by the National Institute of Health.The government has certain rights thereto.

BACKGROUND OF THE DISCLOSURE

The basis of some of the embodiments of the present disclosure is thetrapping of target molecules by a recognition reagent (referred to as areader molecule) tethered to tunneling electrodes, which may be referredto as recognition tunneling (or RT). In a series of earlier disclosures,WO2009/117522A2, WO2010/042514A1, WO2009/117517, WO2008/124706A2, and WO2011/097171, each of which is incorporated herein by reference, systemsand methods are disclosed where nucleic acid bases may be read by usingthe electron tunneling current signals generated as the nucleo-basespass through a tunnel gap consisting of two electrodes functionalizedwith reader molecules. A demonstration of the ability of this system toread individual bases embedded in a polymer was given by Huang et al.¹

RT may be also used to read an amino acid sequence, leading to proteinsequencing in a nanopore/orifice, as set out in WO 2013/116509, entitled“Systems, Apparatuses And Methods For Reading an Amino Acid Sequence”(“the '509 publication”). In the '509 publication, two methods aredisclosed for sequencing proteins based on recognition tunneling. In onemethod, an enzyme coupled to a functionalized orifice or nanopore isused to feed amino acids into a tunnel junction as they are sequentiallycleaved from the end of a protein chain. The second method feeds intactpeptides through a nanopore where the amino acid sequence is read out aseach amino acid residue passes through the tunnel junction. In some ofembodiments of the second approach, the method is simpler and morestraightforward, and may be configured to produce longer sequence reads.

The '509 publication also describes methods and systems for readingnegatively or positively charged peptides using a pair of nanopores—onebiased to attract the positive peptides, the other biased to attract thenegative peptides, and methods and systems where neutral peptides arepulled through a nanopore by electro osmosis. A possible issue with thisapproach is that it requires a separate arrangement for each type of netpeptide charge, positive, negative and neutral. Furthermore, theapproaches yield no information about which end, N or C terminus, of thepeptide enters the nanopore first. Finally, the electrical force on thepeptide varies with the charge on the peptide, so a significant run ofneutral residues or residues of opposite charge to the overall charge ofthe peptide result in no force (or even a reversed force) on segments ofthe peptide. In fact, significant forces may be required to pull thepeptide through the nanopore in order to overcome the tendency of thepeptide to fold.

Accordingly, in view of the above-noted issues, it would be desirable todevelop a method to draw peptides of any charge through a nanopore andto do so in a known orientation (N or C terminus first). In addition, itis desirable to exert a known force pulling on the peptide, independentof its particular charge. By means of protein expression in cells, Niviaet al.³ have engineered proteins by fusing negatively charged peptidesinto their C-terminus in order to drag the protein into the nanopore,regardless of the intrinsic charge on the polymer. This procedure willnot work on the naturally occurring proteins that one would wish tosequence with a nanopore. Accordingly, this disclosure providesprocedures for attaching charged polymers to N termini ofnaturally-occurring proteins.

Additionally, the current dominant method for proteomic analysis is massspectrometry, which typically requires as much as a microgram of protein(corresponding to 0.1 nanomoles of a 10 kD protein), though samples ofseveral mg are required for enrichment to detect low abundance (<5%)modified sites (e.g. phosphorylation). In some RT systems foridentifying amino acids (e.g., the '509 publication), sampleconcentrations of about 10 uM and working volumes of 0.1 ml arerequired, which correspond to a nanomole of sample (i.e., samplerequirements are comparable to mass spectrometry). Since samples aredelivered to the tunnel junction by diffusion, the quantification ofmixed analytes is complicated by differential diffusion and differentialbinding in the junction. Accordingly, it is desirable to have a methodthat delivers samples to the junction in a more deterministic way, anduses lower concentrations of analyte.

At least some of these and other goals may be achieved by at least someembodiments of the present disclosure.

SUMMARY OF SOME OF THE EMBODIMENTS

Accordingly, at least some of the embodiments supported by the presentdisclosure address one and/or another of the issues discussed above.

In some embodiments, a device for analyzing the molecules is providedand includes a first volume of conducting fluid, a second volume ofconducting fluid, an orifice in communication with said first and secondvolumes of fluid, and means for applying an electric potentialdifference between said first and second volumes of fluid. In some suchembodiments, a conjugate product (which may also be referred to asproduct) is provided which comprises charged polymers each havingattached thereto at least one first molecule for analysis, where theproduct carries a predetermined charge greater than the charge on thefirst molecule, and upon dissolving a product in the first volume offluid, the product is directed into the orifice.

In some embodiments, a device for directing a peptide molecule foranalysis into an orifice of an analysis device is provided includes afirst chamber and a second chamber, where each compartment includes anelectrolytic solution. The device also includes a membrane separatingthe second chamber from the first chamber, an orifice provided in themembrane configured to receive and/or pass a product between the firstand the second chambers, a first electrode provided in the firstchamber, and a second electrode provided in the second chamber. Upon theproduct, e.g., a conjugate product comprising a plurality of peptidefragments for analyzing, each fragment being coupled at a correspondingN-termini to a polymeric ion via a cross linker (a polymeric ion being apolymer comprising multiple charges) being dissolved in the electrolytesolution in the first chamber and a bias being applied between the firstand second electrodes, the product is at least pulled into the nanoporeupon the bias of the second electrode being configured to attract thepolymeric ion of the product.

In such embodiments, the polymeric ion comprises repeated negativecharges, and upon the first electrode being biased negative and thesecond electrode being biased positive, the negatively charged polymericions are pulled into the nanopore.

In such embodiments, upon a first electric field of a firstpredetermined amount being established on a first side of the nanoporeand facing the first chamber, the first electric field extends adistance λ from the nanopore, and a second electric field of a secondpredetermined amount is established on a second side of the nanoporefacing the second chamber, the second electric field extending thedistance λ, the first electric field pulls the product into the nanoporefrom the first chamber, and the second electric field pulls the productout of the nanopore into the second chamber.

In such embodiments, the bias may be configured such that the productdoes not fold. In addition, the plurality of peptide fragments may bebetween about 2 and about 60 peptides.

In some embodiments, a mass spectrometer configured to identify thepeptide fragments may be included.

In some embodiments, the plurality of peptides may be functionalized bymodifying the α-N-terminus with succinic anhydride.

In some embodiments, a pair of orifice electrodes in communication withthe orifice may be provided, where an AC voltage of at least 1 kHz infrequency is applied between the orifice electrodes.

In some embodiments, the presence of a molecule in the orifice may bedetected by means of non-linear processing of the AC current signal.

In some embodiments, an electronic circuit for controlling the values ofbias applied between the first electrode and the orifice electrodes andthe second electrode and the orifice electrodes, where the circuitreceives input from a signal generated by the orifice electrodes. Thevoltage applied between the orifice electrodes may comprise an AC and aDC component.

In some embodiments, a method for generating a product containing aplurality of peptide fragments for analysis is provided, and may includedigesting a peptide having a plurality of lysine residues with Lys-C,trypsin, or trypsin/Lys-C, resulting in a plurality of peptidefragments, each having an α-N-termini, wherein each peptide fragmentincludes a lysine at their C termini, reacting an alkyne modifiedsuccinic anhydride, 3-(2-propynyl)succinic anhydride in a sodium acetatebuffered solution at a predetermined pH to selectively add alkynes tothe α-N-termini of the peptides, and attaching an azide terminatedpolymeric ion to each peptide by the Cu (I) catalyzed reaction of azideand alkyne.

In some embodiments, a method for directing peptide fragments of aprotein into an orifice of a protein sequencing apparatus/device isprovided and includes providing an apparatus/device according to any ofthe disclosed embodiments, digesting a protein for sequencing withendoproteinase Lys-C to produce a plurality of peptide fragments,reacting the fragments with 3-(2-propynyl)succinic anhydride at apredetermined pH in a sodium acetate buffer to produce alkyne-modifiedpeptides, reacting a polyion polymer terminated with an azide with thealkyne-modified peptides in the presence of Cu(I), resulting in aproduct of polymer and allcyne-modified peptide, purifying the producton a size exclusion column, and placing the product in the first chamberof apparatus/device, where the second chamber is biased positively todraw the product through the orifice.

In some embodiments, a method of directing a polymer molecule intoand/or through an orifice of a sequencing device is provided andincludes attaching a charged polymer to one end of a polymer to beanalyzed, resulting in a product, placing the product into a conductingsolution in communication with an orifice, and applying an electricalbias across the orifice such that the charged polymer of the product ispulled into and optionally through the orifice, thereby pulling thepolymer to be analyzed into and optionally through the orifice.

In such embodiments:

the polymer for analysis may be a protein;

the polymer for analysis may be a peptide; and

the polymer molecule may be an oligosaccharide.

In some embodiments, attachment between a charged polymer and thepolymer for analysis is made via a succinic anhydride reaction with aterminal amine of a protein or peptide. The charged polymer may be apolymer of phosphate diesters

In some embodiments, entry of the charged polymer into the orificetriggers control of the bias applied across the orifice.

In some embodiments, the polymer for analysis (i.e., an analyte) istethered to a charged tail, such that transport into the RT junction maybe dominated by the charged tail component (e.g., and not the propertiesof the analyte itself). Additionally, in some embodiments, operating theRT apparatus in a low salt (e.g., ≦0.2M) solution, significant countrates can be obtained with sample concentrations as low as a fewpicomolar (for example). Thus, for example, in 0.1 ml of solution, justa few femtomoles of sample are required, a significant improvement onmass spectroscopy (for example). Furthermore, because transport to theRT junction is deterministic (according to some embodiments), thecontents of the mixture can be quantified just by counting the varioustypes of single molecule signal. Accordingly, in such embodiments, thisremoves the need to enrich samples which, in mass spectrometry, can leadto as much as 20 mg (2 μmoles of a 10 kD protein) of sample beingrequired.

In some embodiments, a method of characterizing modifications of anamino acid or protein is provided which may comprise classifyingrecognition tunneling signals generated by the sequential passage ofmolecules through a recognition tunneling junction according to whethera trained machine-learning algorithm determines each signal as a normalprotein of amino acid, or as a post-translationally modified amino acid,wherein determining comprises counting the number of each type of posttranslational modification, and unmodified molecules. The method mayalso include calculating the fraction of molecules with each posttranslational modification determined.

In some embodiments, a series of new compounds of polyphosphate diesterare presented, as set out in FIG. 5. To that end, in some suchembodiments, a compound of formula (I) is presented:

wherein m may be any of 1-6, k may be any of 1-6, and n may be any of1-300.

In some embodiments, a compound of formula (II) is presented

wherein m may be any of 1-6, n may be any of 1-300, and B may comprisean organic group.

These and some of the many other embodiments taught by the presentdisclosure will become even more evident with reference to the drawingsincluded with the present application (a brief description which isprovided below), and the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration showing how a charged polymer tethered to apeptide of arbitrary charge is used to feed the peptide into a nanoporeaccording to some embodiments of the present disclosure.

FIG. 2 is an illustration showing regions of high electric fields at theentry and exit of the nanopore and how they interact with the chargedpolymer, according to some embodiments of the present disclosure.

FIG. 3 illustrates selective functionalization of the N-terminus of thepeptide fragments according to some embodiments of the disclosure, whereselectivity may be enhanced by digesting the protein into fragmentscontaining only one lysine residue.

FIG. 4 illustrates covalent conjugation of the functionalized proteinfragments with a polyermeric ion according to some embodiments of thepresent disclosure.

FIG. 5 illustrates examples of polymeric ions suitable for conjugationwith functionalized peptides according to some embodiments of thepresent disclosure.

FIG. 6 illustrates examples of a polycation, including azidopolyethylenimine and azido polylysine, according to some embodiments ofthe present disclosure.

FIG. 7 a-d, illustrate blockade signals from buffer alone (a), DNA(dT₂₀) alone (b), angiotensin alone (c), and upon the DNA-angiotensinconjugate being added, frequent blockade signals are observed again (d),according to some embodiments of the present disclosure.

FIGS. 8 a-c, illustrate (a) scatter plot of blockade time and blockadeamplitude for ssDNA alone (gray) and the angiotensin-DNA conjugate(black); (b) and (c) show examples of signals (curved traces) as fittedto obtain amplitudes and widths (block traces), according to someembodiments of the present disclosure.

DETAILED DESCRIPTION OF SOME OF THE EMBODIMENTS

It is an object of at least some of the embodiments of the presentdisclosure to provide a system and method to draw peptides of any chargeinto and/or through a nanopore (the term nanopore and orifice usedinterchangeably throughout this disclosure) and to do so in a knownorientation (N or C terminus first). In addition, it is an abject of atleast some of the embodiments of the present disclosure to exert a knownforce to pull on the peptide, independent of its particular charge.These and other goals are achieved by at least some of the embodimentsof the present disclosure.

Accordingly, some of the embodiments of the present disclosure areoutlined schematically in FIG. 1. As shown, in some embodiments, peptidefragments 1, obtained by enzymatic digestion of the protein to besequenced (for example), are coupled at their N-terminus by means of across linker 2 to a polymeric ion 3 chosen to carry much larger chargethan any of the protein fragments. Thus, if a typical fragment size isbetween about 10 to about 100 amino acids in length, the polymeric ion 3would be at several tens to several hundred of units in length, so as tobe longer than the peptide fragments. With the length (and hence charge)of the polymeric ion being greater than the length (and thus maximumpossible charge) of the peptide fragment, the net charge of the systemof peptide fragment 1, cross linker 2, and polymeric ion 3 is determinedby the polymeric ion. When the conjugate (1-2-3) is dissolved in anelectrolyte solution 4 contained in two compartments (labeled cis andtrans) separated by a nanopore 5 in a membrane 6 (that separates the twocompartments), and a bias V1 (7) is applied between a referenceelectrode in the cis compartment, R1 (8) and a reference electrode inthe trans compartment, R2 (9), the conjugate (1-2-3) will be pulled intothe nanopore if the bias of R2 (9) is such as to attract the polymericion. For example, if the polymeric ion consists of repeated negativecharges, such as phosphate ions, then R1 will be biased negative, and R2biased positive to pull the negative polymeric ion into the pore.

In some embodiments, the motion of the peptide section to be sequenceddepends on the pore geometry, as illustrated in FIG. 2. Specifically,regions of high electric field are confined to the ends of the nanoporeand are shown extending a distance from the end of the nanopore. λ issomewhat salt-dependent, varying between about 10 nm at 0.01M salt and 5nm at 1 M salt (see calculation shown in FIG. S22 of Peng et al.⁴).Accordingly, in some embodiments, the charged polymer is pulled into thepore from the cis side by the high field region on this side, 10, andpulled out again by the high field region on the trans side, 11. Thus,in such embodiments, the polymeric ion 3 is drawn into the pore and thenout again. In some embodiments, the force on the polymer is independentof pore and polymer length, depending only on the potential differenceapplied across the pore. For example, for a DNA polymeric ion, the forcegenerated is 0.24 pN/mV (see paper by Keyser et al.⁵). Since the forceto unfold a single IgG domain (an example of a tightly folded protein)is in the range of about 5 to about 40 pN, a bias across the pore ofjust 170 mV is adequate to overcome peptide folding, pulling the peptideinto the pore as a linear structure (according to some embodiments).

In some embodiments, the pore length, L, is typically about 20 nm, longenough to contain about 40 peptide residues (of about 0.5 nmresidue-to-residue spacing). The polymeric ion 3 thus controls themotion of the peptide over a distance of about L+λ. For example, in thecase where L is 20 nm and λ=5 nm, together equal to 25 nm correspondingto about 50 amino acid residues. Once the polymeric ion has passed outof the high field region, subsequent transport (of an uncharged peptide)is dominated by diffusion, a much slower process. Thus, in a typicalnanopore with dimensions given above, active electric field control oftranslocation is limited to no more than about 50 amino acid residues.Accordingly, in some embodiments, this is adequate for “Bottom Up”protein identification by mass spectrometry.⁶ In such a case, thepolymeric ion 3 is at least 100 units in length to guarantee that thetotal charge of the assembly was completely dominated by the polymericion. In some embodiments, a longer nanopore channel length (L in FIG. 2)and a longer polymeric ion allow longer peptide fragments to be pulledthrough the pore. For example, the largest IgG domain is about 110 aminoacids, requiring a pore length of about 50 nm and a polymeric ion ofabout 200 charged units in length.

In order to connect a polymeric ion to a peptide fragment, it isnecessary to selectively functionalize an end of the peptide withoutmodifying the side chains. This can be done using modification of theα-N-terminus with succinic anhydride. When this reaction is carried outis sodium acetate buffer at pH 7.6, the N-terminus may be modified whilelysines (the only other charged primary amines among the naturallyoccurring amino acid residues) are left unmodified.⁷ It is helpful toincrease the density of accessible N-termini relative to lysine residuesbecause the probability of an unwanted reaction with the primary aminein lysine residues will increase as the number of lysine residues in aprotein molecule. Accordingly, the specificity of the reaction isincreased by cutting the protein into fragments containing only onelysine residue each. This is readily achieved using endoproteinase suchas Lys-C.⁷

A coupling process is illustrated in FIGS. 3 and 4. As an example, apeptide containing two lysine residues (“K”) is shown (20). The thicksolid bar represents other residues in the peptide. Digestion with Lys-C(21) produces three fragments (22), only two of which contain one lysinein their respective sequences and the rest contains no lysine. Althoughpresenting three N-termini (23), it reduces the possibility for unwantedreactions from lysines. An alkynylated succinic anhydride,3-(2-propynyl)succinic anhydride (24) is used to selectively addallcynes (26) to the N-termini of the peptides (25) in the sodiumacetate buffered solution at pH 7.6. An azide terminated polymeric ionis attached to the peptide by the Cu (I) catalyzed reaction of azide andethyne well known in the art (i.e., “click” chemistry), which isillustrated in FIG. 4. Here the polymeric ion is shown as a genericpolyphosphate diester (31) terminated with an azide moiety (32).Reaction with the alykynated peptide 25 fragments in the presence of aCu (I) catalyst (33) produces the concatenated molecule (34). In thiscase, the peptide is coupled to the polymeric ion at its N-terminus, andthis is the end that will be threaded into the nanopore first, achievingthe goal of orienting the peptide for a sequence read.

Examples of suitable polyanions are shown in FIG. 5. The simplestdiester consists of a chain of phosphates (41) linked by k repeats ofmethylene groups (k=1 to 6 are reasonable values, both easy tosynthesize and soluble in aqueous buffer). The polyanion is terminatedin an azide 42 linked by m repeated methylene units where m=1 to 6. Inanother embodiment, the polymer repeat (43) includes a sugar with anorganic residue B (44) (for example DNA where B could be the T base).Using a simple phenol group for B may ensure no hydrogen bonding, andthus, no recognition tunneling signals. However, the use of a DNA basewould generate recognition tunneling signals ahead of the peptidesignals, and may prove useful for calibrating the readout. The polymeris again terminated in an azide (42). By the same token, a polycationsuch as azido polyethylenimine (45) and azido polylysine (46) (FIG. 6)can be used for the translocation. In this case, the polarity of theelectrodes would be reversed so that the cargo to be analyzed would bepulled towards the negative electrode.

In some embodiments, the method for sequencing a protein includes two ormore of the following steps (and in some embodiments, all steps):

-   -   the protein to be sequenced is digested with endoproteinase        Lys-C, and/or trypsin;    -   the resulting fragments are reacted with 3-(2-propynyl)succinic        anhydride at pH 7.6 or below in a sodium acetate buffer;    -   a polyion polymer terminated with an azide reacts with the        alkyne-modified peptides in the presence of Cu(I);    -   the conjugate product (polymer ion+peptide) is purified on a        size exclusion column; and    -   the polyion labeled peptide mix is placed in the cis chamber of        a nanopore apparatus/device, and the trans chamber is biased        positively to draw the concatenated polymers through the        nanopore;

In some embodiments, the tunneling signal generated by the polymeric ionprovides an advance notice of the arrival of the following peptide chainand can be used to activate control circuitry as described below. Itwill be recognized that a similar approach could be used to pull otherpolymers (such as poly saccharides) through nanopores for sequencing. Inthe case of polysaccharides, specific terminal functionalization at aterminal —OH group may be unlikely to be successful because of the —OHgroups in the sugars. However, the additional step of separatingend-functionalized molecules by chromatography may be used to addressthis.

In some embodiments, azidoacetic anhydride can be used as a substituentof 3-(2-propynyl)succinic anhydride. Preparation of azidoaceticanhydride (Scheme 1):

N,N′-Dicyclohexylcarbodiimide (DCC, 204 mg, 0.98 mmol) was added to asolution of 2-azidoacetic acid (200 mg, 1.9 mmol) in anhydroustetrahydrofuran. The mixture was stirred for 2 hr and 15 min, filtered.The filtrate was concentrated by rotary evaporation, furnishingazidoacetic anhydride (150 mg, 42%) as a colorless liquid. ¹H NMR (400MHz, CDCl₃): δ=3.85 (s, 4H); ¹³C NMR (100 MHz, CDCl₃): δ=168.3, 50.0ppm.

Example: reaction of azidoacetic anhydride with a peptide bearing onelysine (Scheme 2).

A solution of azidoacetic anhydride (1 mM, 15 μL) in acetonitrile wasadded to the peptide solution (50 μM, 15 μL) in a sodium acetate buffer(50 mM, pH 6.7) in an eppendorf tube at 0C. Before the addition, bothazidoacetic anhydride and peptide solutions were cooled at 0° C. for 10min. After 30 min, MALDI-TOF mass spectrometry showed that the peptidestarting material was consumed and a new product produced. The productwas characterized as an azidoacetyl mono substituted peptide by MALDImass: m/z (M+H) calculated: 1083.12; found: 1083.33.

Example: Control study (scheme 3).

To demonstrate that the reaction selectively takes place at the Nterminus, the same peptide with N-terminus blocked by an azidoacetylgroup reacted with azidoacetic anhydride under the exactly sameconditions. MALDI mass spectrometry showed that no reaction took placeeven after 40 min. This confirmed that azidoacetylation was occurringonly at the N-telminus of the peptide.

Example: Reaction of azidoacetic anhydride with a peptide sequencehaving three lysines (Scheme 4):

A solution of azidoacetic anhydride (1 mM, 15 μL) in acetonitrile wasadded to the peptide solution (50 μM, 15 μL) in a sodium acetate buffer(50 mM, pH 6.7) in an eppendorf tube at 0° C. Before the addition, bothazidoacetic anhydride and peptide solutions were cooled at 0° C. for 10mM. After 20 mM, MALDI-TOF mass spectrometry showed that the peptidestarting material was consumed and a new product produced. The productwas characterized as an azidoacetyl mono substituted peptide by MALDImass: m/z (M+H) calculated: 1097.17; found: 1097.45.

Example: Control Study (Scheme 5)

The same peptide with N-terminus blocked by an acetyl group reacted withazidoacetic anhydride under the exactly same conditions. MALDI massspectrometry showed that no reaction took place even after 45 mM. Thisconfirmed that azidoacetylation was occurring only at the N-terminus ofthe peptide.

Example: Synthesis of an angiotensin I and PolyT₂₀ conjugate.

A. Modification of Angiotensin I Using Azidoacetic Anhydride (Scheme 6):

Angiotensin I (50 μM, 15 μL) in a sodium acetate buffer (50 mM, pH 6.7)was added to a solution of azidoacetic anhydride (1 mM, 15 μL) inacetonitrile in an eppendorf tube at 0° C. Before the addition, bothazidoacetic anhydride and peptide solutions were cooled at 0° C. for 10min. After 15 min, MALDI mass spectrometry showed that the angiotensinwas consumed and a new product produced. The solvent was removed usingSpeedVac and the residue was redissolved in of TEAA buffer (pH 7, 10μL). The product was purified by reverse phase HPLC in a Zorbax EclipsePlus C18 column (4.6×150 mm, particle size 5 μm) with an acetonitrilegradient of 0 to 70% over a period of 30 min (solvent A: 0.1 M TEAAbuffer, pH 7.0; solvent B: acetonitrile). The major fraction at theretention time of 16.5 min was collected and lyophilized. MALDI-TOF massspectrometry showed that it was an azidoacetyl mono substituted peptideproduct. The mlz (M+H) calculated: 1379.54; found: 1379.66.

B. Modification of Poly T₂₀ by DBCO (Scheme 7):

An oligonucleotide Poly T₂₀ with a C12 amino Modifier at its 5′ end (1mM, 10 μL) in water was added into a Phosphate buffer (30 μL, pH 8.5).To this solution was added a solution of DBCO-NHS ester in DMSO (15 mM,40 μL), which was shaken for 30 min at room temperature, followed byanother addition of the DBCO-NHS ester (40 μL). The reaction mixture wasshaken for additional 1.5 h. The product was purified by reverse phaseHPLC in a Zorbax Eclipse Plus C18 column (4.6×150 mm, particle size 5μm) with an acetonitrile gradient of 0 to 60% over a period of 25 min(solvent A: 0.1 M TEAA buffer, pH 7.0; solvent B: acetonitrile) andcharacterized by MALDI-MS. The m/z (M+H) calculated 6667.23; found:6676.34.

C. Coupling of Poly T₂₀ to Angiotensin I by the Click Reaction (Scheme8).

A solution of the azido modified peptide (30 μM, 15 μL)) in the TEAAbuffer (50 mM, pH 7) was added to a solution of the DBCO functionalizedPolyT₂₀ (5 μM, 10 μL,)) in the TEAA buffer (50 mM, pH 7), shaken at roomtemperature for 3 hours. The product was purified employing reversephase HPLC in a Zorbax Eclipse Plus C18 column (4.6×150 mm, particlesize 5 μm) with an acetonitrile gradient of 0 to 60% over a period of 25min (solvent A: 0.1 M TEAA buffer, pH 7.0; solvent B: acetonitrile). Theproduct had a retention time of 5.4 min. and characterized by MALDI-MS(m/z (M+H) of the product calculated: 7994.12; found: 7996.15). Afterlyophilization, the product was given as a white powder.

Demonstration of translocation of the peptide-DNA complex through ananopore.

FIGS. 7 a-d show example ion-current blockade signals from samples whichdemonstrate how conjugation_(—) of the angiotensin peptide with anegatively charged PolyT₂₀ ssDNA results in the peptide being pulledthrough a 3 nm diameter nanopore, according to some embodiments. FIG. 7a shows a control signal obtained in the salt solution alone (0.4 M KClwith 10 mM Tris buffer, pH=7.0). The bias across the nanopore was 400mV. On adding 1 μM PolyT₂₀ ssDNA, frequent current blockades areobserved (FIG. 7 b). On rinsing, the signal returns to the featurelesscontrol signal (see FIG. 7 a). When 1 ‘μM angiotensin is added,blockades are observed only occasionally (FIG. 7 c). However, when theconjugated product (Scheme 8, above) is added (concentration about 1μM), frequent blockade signals are observed (FIG. 7 d). Signals from theconjugated product are distinctive, having a slightly smaller blockadeamplitude and considerably longer duration than blockades owing to thessDNA alone (FIG. 8 a). Examples of fitted blockade signals are givenfor the ssDNA in FIG. 8 b and for the angeotensin-DNA conjugate in FIG.8 c.

Thus, in some embodiments, a peptide or amino acid may be linked to acharged tail. In such embodiments, a tail comprising charged amino acids(see also, Scheme 8, infra, a charged tail linked to a single-strandedDNA, e.g., a 20 nucleotide oligothymine, T₂₀. Thus, in some embodiments,neutral analytes can be added to a threader molecule carryingsubstantial charge (e.g., 20 negative charges), so long as a terminalamine is available for the attachment chemistry (for example).

Accordingly, this may bring about a significant reduction in the amountof analyte, and concentration of analyte, required. While neutralmolecules, such as polyethylene glycol and oligosaccharides, have beentranslocated through nanopores, mM concentrations were necessary toachieve a count rate of a few counts per second. In sharp contrast, andfor example, in low salt concentrations (e.g.,≦0.2M), similar countrates were achieved for small DNA molecules at concentrations as low as,for example, 4 pM. This may be because the electric field gradient nearthe nanopore can collect charged molecules from a large volume of samplespace.

Example: an analyte containing a terminal amine, is modified withazidoacetic anhydride to produce an azide termination. This molecule isthen reacted with a charged polymer (a DNA oligomer in the preferredembodiment) that is coupled to DBCO (scheme 8). The product of thisreaction is then placed in a chamber on one side (by convention, the cisside) of a nanopore articulated with a recognition-tunneling junction ina concentration that can be as low as 1 pM (for example). A positivebias of +300 to +400 mV with respect to a reference electrode on the cisside is applied to a reference electrode on the opposite (trans) side.The charged polymer is carried through the nanopore by electrophoresis,thereby pulling the analyte with it. The recognition-tunneling junctionmay then generate a signal characteristic of the charged polymer, whichmay be followed by a signal characteristic of the analyte. Thus,according to some embodiments (an example of which is immediatelyabove), a neutral analyte, which would normally have to present at mMconcentration to be translocated at rate >1 count per second, may betranslocated and analyzed at a count rate >count per second withconcentrations as low as 1 pM.

Various implementations of the embodiments disclosed, in particular atleast some of the processes discussed, may be realized in digitalelectronic circuitry, integrated circuitry, specially designed ASICs(application specific integrated circuits), computer hardware, firmware,software, and/or combinations thereof. These various implementations mayinclude implementation in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which may be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device.

Such computer programs (also known as programs, software, softwareapplications or code) include machine instructions for a programmableprocessor, for example, and may be implemented in a high-levelprocedural and/or object-oriented programming language, and/or inassembly/machine language. As used herein, the term “machine-readablemedium” refers to any computer program product, apparatus and/or device(e.g., magnetic discs, optical disks, memory, Programmable Logic Devices(PLDs)) used to provide machine instructions and/or data to aprogrammable processor, including a machine-readable medium thatreceives machine instructions as a machine-readable signal. The term“machine-readable signal” refers to any signal used to provide machineinstructions and/or data to a programmable processor.

To provide for interaction with a user, the subject matter describedherein may be implemented on a computer having a display device (e.g., aCRT (cathode ray tube) or LCD (liquid crystal display) monitor and thelike) for displaying information to the user and a keyboard and/or apointing device (e.g., a mouse or a trackball) by which the user mayprovide input to the computer. For example, this program can be stored,executed and operated by the dispensing unit, remote control, PC,laptop, smart-phone, media player or personal data assistant (“PDA”).Other kinds of devices may be used to provide for interaction with auser as well; for example, feedback provided to the user may be any formof sensory feedback (e.g., visual feedback, auditory feedback, ortactile feedback); and input from the user may be received in any form,including acoustic, speech, or tactile input.

Certain embodiments of the subject matter described herein may beimplemented in a computing system and/or devices that includes aback-end component (e.g., as a data server), or that includes amiddleware component (e.g., an application server), or that includes afront-end component (e.g., a client computer having a graphical userinterface or a Web browser through which a user may interact with animplementation of the subject matter described herein), or anycombination of such back-end, middleware, or front-end components. Thecomponents of the system may be interconnected by any form or medium ofdigital data communication (e.g., a communication network). Examples ofcommunication networks include a local area network (“LAN”),-a wide areanetwork (“WAN”), and the Internet.

The computing system according to some such embodiments described abovemay include clients and servers. A client and server are generallyremote from each other and typically interact through a communicationnetwork. The relationship of client and server arises by virtue ofcomputer programs running on the respective computers and having aclient-server relationship to each other.

Any and all references to publications or other documents, including butnot limited to, patents, patent applications, articles, webpages, books,etc., presented anywhere in the present application, are hereinincorporated by reference in their entirety.

Although a few variations have been described in detail above, othermodifications are possible. For example, any logic flows depicted in theaccompanying figures and/or described herein do not require theparticular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of at least someof the following exemplary claims.

As noted elsewhere, these embodiments have been described forillustrative purposes only and are not limiting. Other embodiments arepossible and are covered by the disclosure, which will be apparent fromthe teachings contained herein. Thus, the breadth and scope of thedisclosure should not be limited by any of the above-describedembodiments but should be defined only in accordance with claimssupported by the present disclosure and their equivalents. Moreover,embodiments of the subject disclosure may include methods, systems andapparatuses/devices which may further include any and all elements fromany other disclosed methods, systems, and devices, including any and allelements corresponding to translocation control. In other words,elements from one or another disclosed embodiments may beinterchangeable with elements from other disclosed embodiments. Inaddition, one or more features/elements of disclosed embodiments may beremoved and still result in patentable subject matter (and thus,resulting in yet more embodiments of the subject disclosure). Also, someembodiments correspond to systems, devices and methods whichspecifically lack one and/or another element, structure, and/or steps(as applicable), as compared to teachings of the prior art, andtherefore represent patentable subject matter and are distinguishabletherefrom.

REFERENCES

-   1 Huang, S. et al. Identifying single bases in a DNA oligomer with    electron tunneling. Nature Nanotechnology 5, 868-873 (2010).-   2 Liang, F., Li, S., Lindsay, S. & Zhang, P. Synthesis,    Physicochemical Properties, and Hydrogen Bonding of    4(5)-Substituted-1H-imidazole-2-carboxamide, A Potential Universal    Reader for DNA Sequencing by Recognition Tunneling. Chemistry—a    European Journal 18, 5998-6007 (2012).-   3 Nivaia, J., Marks, D. B. & Akeson, M. Unfoldase-mediated protein    translocation though an alpha-hemolysin pore. Nature Biotechnol. 31,    247-250 (2013).-   4 Pang, P., He, J., Park, J. H., Krstic, P. S. & Lindsay, S. Origin    of Giant Ionic Currents in Carbon Nanotube Channels. ACS Nano 5,    7277-7283 (2011).-   5 Keyser, U. F. et al. Direct force measurements on DNA in a    solid-state nanopore. Nature Physics 2, 473-477 (2006).-   6 Thakur, S. S. et al. Deep and Highly Sensitive Proteome Coverage    by LC-MS/MS Without Prefractionation. Molecular & Cellular    Proteomics 10, M110.003699-003691-M003110.003699-003699. (2011).-   7 Koehler, C. J., Arntzen, M. Ø., Strozynski, M., Treumann, A. &    Thiede, B. Isobaric Peptide Termini Labeling Utilizing Site-Specific    N-Terminal Succinylation. Analytical Chemistry 83, 4775-4781 (2011).

1. A device for analyzing molecules comprising: a first volume ofconducting fluid; a second volume of conducting fluid; an orifice incommunication with said first and second volumes of fluid; and means forapplying an electric potential difference between said first and secondvolumes of fluid, wherein: a product comprising charged polymers isprovided, each charged polymer having attached thereto at least onefirst molecule for analysis, the first molecule having an amine terminusto which the charged polymer is attached, the product carries apredetermined charge greater than the charge on the first molecule, andupon dissolving the product in the first volume of fluid, the product isdirected into the orifice.
 2. A device for directing a peptide moleculefor analysis into an orifice of an analysis device, the systemcomprising: a first chamber and a second chamber, wherein eachcompartment includes an electrolytic solution; a membrane separating thesecond chamber from the first chamber; an orifice provided in themembrane configured to receive and/or pass the product between the firstand the second chambers; a first electrode provided in the firstchamber; and a second electrode provided in the second chamber; whereina product is provided comprising a plurality of peptide fragments foranalyzing, each fragment being coupled at a corresponding N-termini to apolymeric ion via a cross linker, and upon the product being dissolvedin the electrolyte solution in the first chamber and a bias beingapplied between the first and second electrodes, the product is at leastpulled into the nanopore upon the bias of the second electrode beingconfigured to attract the polymeric ion of the product.
 3. The device ofclaim 2, wherein upon the polymeric ion comprising repeated negativecharges, and upon the first electrode being biased negative and thesecond electrode being biased positive, the negatively charged polymericions are pulled into the nanopore.
 4. The device of claim 2, wherein afirst electric field of a first predetermined amount is established on afirst side of the nanopore facing the first chamber, the first electricfield extending a distance λ from the nanopore, and a second electricfield of a second predetermined amount is established on a second sideof the nanopore facing the second chamber, the second electric fieldextending the distance λ, wherein the first electric field pulls theproduct into the nanopore from the first chamber, and the secondelectric field pulls the product out of the nanopore into the secondchamber.
 5. The device of claim 2, wherein the bias is configured suchthat the product does not fold.
 6. The device of claim 2, wherein theplurality of peptide fragments comprise between about 2 and about 60peptides.
 7. The device of claim 2, further comprising a massspectrometer configured to identify the peptide fragments.
 8. The deviceof claim 2, wherein the plurality of peptides are functionalized bymodifying the N-terminus with succinic anhydride.
 9. The device of claim2, further comprising a pair of orifice electrodes in communication withthe orifice, wherein an AC voltage of at least about 1 kHz in frequencyis applied between the orifice electrodes.
 10. The device of claim 9,wherein the presence of a molecule in the orifice is detected by meansof non-linear processing of the AC current signal.
 11. The device ofclaim 9, further comprising an electronic circuit for controlling thevalues of bias applied between the first electrode and the orificeelectrodes and the second electrode and the orifice electrodes, whereinthe circuit receives input from a signal generated by the orificeelectrodes.
 12. The device of claim 9, wherein the voltage appliedbetween the orifice electrodes comprises an AC and a DC component. 13.The device of claim 2, further comprising the electrolytic solution inthe first chamber, wherein the electrolytic solution comprises a saltsolution of less than about 1M concentration.
 14. The device of claim13, further comprising a salt solution in the second chamber.
 15. Thedevice of claim 2, wherein the plurality of peptides are functionalizedby modifying the N-terminus with azidoacetic anhydride.
 16. (canceled)17. A method for directing peptide fragments of a protein into anorifice of a protein sequencing apparatus/device, the method comprising:providing the system of claim 2; digesting a protein for sequencing withendoproteinase Lys-C to produce a plurality of peptide fragments;reacting the fragments with 3-(2-propynyl)succinic anhydride at apredetermined pH in a buffered solution to produce alkyne-modifiedpeptides; reacting a polyion polymer terminated with an azide with thealkyne-modified peptides in the presence of Cu(I), resulting in aconjugate product of polymer and peptide; purifying the conjugateproduct on a size exclusion column; placing the conjugate in the firstchamber of the system of claim 2, wherein the second chamber is biasedpositively to draw the conjugate product through the orifice. 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. A methodfor directing peptide fragments of a protein into an orifice of aprotein sequencing apparatus/device, the method comprising: providingthe system of claim 2; digesting a protein for sequencing withendoproteinase Lys-C, trypsin, or Lys-C/trypsin to produce a pluralityof peptide fragments; reacting the fragments with azidoacetic anhydrideat a predetermined pH in a sodium acetate or other buffered solution toproduce alkyne-modified peptides; reacting a polyion polymer terminatedwith a cyclooctyne or its derivative with the azide-modified peptides inthe absence of Cu(I), resulting in a conjugate product of polymer andpeptide; purifying the conjugate product on a size exclusion column;placing the conjugate in the first chamber of the system of claim 2,wherein the second chamber is biased positively to draw the conjugateproduct through the orifice.
 28. (canceled)
 29. (canceled)